Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30?years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.     

An electronic analog of synthetic genetic networks

An electronic analog of a synthetic genetic network known as the repressilator is proposed. The repressilator is a synthetic biological clock consisting of a cyclic inhibitory network of three negative regulatory genes which produces oscillations in the expressed protein concentrations. Compared to previous circuit analogs of the repressilator, the circuit here takes into account more accurately the kinetics of gene expression, inhibition, and protein degradation. A good agreement between circuit measurements and numerical prediction is observed. The circuit allows for easy control of the kinetic parameters thereby aiding investigations of large varieties of potential dynamics.     

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j-, respectively). Although unlike eIF3j+, eIF3j- does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2*GTP*Met-tRNA(i)Met ternary complex, eIF3j-/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j- nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.     

The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH

Eukaryotic initiation factor eIF1 and the functional C-terminal domain of prokaryotic initiation factor IF3 maintain the fidelity of initiation codon selection in eukaryotes and prokaryotes, respectively, and bind to the same regions of small ribosomal subunits, between the platform and initiator tRNA. Here we report that these nonhomologous factors can bind to the same regions of heterologous subunits and perform their functions in heterologous systems in a reciprocal manner, discriminating against the formation of initiation complexes containing codon-anticodon mismatches. We also show that like IF3, eIF1 can influence initiator tRNA selection, which occurs at the stage of ribosomal subunit joining after eIF5-induced hydrolysis of eIF2-bound GTP. The mechanisms of initiation codon and initiator tRNA selection in prokaryotes and eukaryotes are therefore unexpectedly conserved and likely involve related conformational changes induced in the small ribosomal subunit by factor binding. YciH, a prokaryotic eIF1 homologue, could perform some of IF3's functions, which justifies the possibility that YciH and eIF1 might have a common evolutionary origin as initiation factors, and that IF3 functionally replaced YciH in prokaryotes.     

The eIF1A solution structure reveals a large RNA-binding surface important for scanning function

The translation initiation factor eIF1A is necessary for directing the 43S preinitiation complex from the 5' end of the mRNA to the initiation codon in a process termed scanning. We have determined the solution structure of human eIF1A, which reveals an oligonucleotide-binding (OB) fold and an additional domain. NMR titration experiments showed that eIF1A binds single-stranded RNA oligonucleotides in a site-specific, but non-sequence-specific manner, hinting at an mRNA interaction rather than specific rRNA or tRNA binding. The RNA binding surface extends over a large area covering the canonical OB fold binding site as well as a groove leading to the second domain. Site-directed mutations at multiple positions along the RNA-binding surface were defective in the ability to properly assemble preinitiation complexes at the AUG codon in vitro.     

A distinct class of internal ribosomal entry site in members of the Kobuvirus and proposed Salivirus and Paraturdivirus genera of the Picornaviridae

The 5'-untranslated regions (5' UTRs) of picornavirus genomes contain an internal ribosomal entry site (IRES) that promotes the end-independent initiation of translation. Picornavirus IRESs are classified into four structurally distinct groups, each with different initiation factor requirements. Here, we identify a fifth IRES class in members of Kobuvirus, Salivirus, and Paraturdivirus genera of Picornaviridae: Aichi virus (AV), bovine kobuvirus (BKV), canine kobuvirus (CKoV), mouse kobuvirus (MKoV), sheep kobuvirus (SKV), salivirus A (SV-A), turdivirus 2 (TV2), and TV3. The 410-nucleotide (nt)-long AV IRES comprises four domains (I to L), including a hairpin (L) that overlaps a Yn-Xm-AUG (pyrimidine tract/spacer/initiation codon) motif. SV-A, CKoV, and MKoV also contain these four domains, whereas BKV, SKV, and TV2/TV3 5' UTRs contain domains that are related to domain I and equivalent to domains J and K but lack an AV-like domain L. These IRESs are located at different relative positions between a conserved 5'-terminal origin of replication and divergent coding sequences. Elements in these IRESs also occur elsewhere: domain J's apical subdomain, which contains a GNRA tetraloop, matches an element in type 1 IRESs, and eIF4G-binding motifs in domain K and in type 2 IRESs are identical. Other elements are unique, and their presence leads to unique initiation factor requirements. In vitro reconstitution experiments showed that like AV, but in contrast to other currently characterized IRESs, SV-A requires the DExH-box protein DHX29 during initiation, which likely ensures that the initiation codon sequestered in domain L is properly accommodated in the ribosomal mRNA-binding cleft.